INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 34: 1219-1224, 2014
Transforming growth factor β regulates β-catenin expression in lung fibroblast through NF-κB dependent pathway JIAN LI1, GANG WANG2 and XIA SUN3 1
Jinan Centre for Disease Control and Prevention, Jinan, Shandong 250001; 2Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou, Jiangsu 223002, P.R. China; 3 Department of Pediatrics, The University of Chicago, Chicago, IL 60637, USA Received March 7, 2014; Accepted August 8, 2014 DOI: 10.3892/ijmm.2014.1916
Abstract. β-catenin contributes to the pathogenesis of lung fibrosis. However, the expression of β -catenin in fibroblasts under fibrotic conditions has not been studied. We investigated the expression of β-catenin in lung fibroblasts from bleomycin (BLM)‑challenged mice and human lung fibroblasts treated with transforming growth factor β (TGF-β) or lysophosphatidic acid (LPA) by western blot analysis. The result showed that the expression of β-catenin was significantly increased in lung fibrotic foci and lung fibroblasts from bleomycin‑challenged mice. TGF- β stimulated β -catenin expression and induced differentiation in human lung fibroblasts in vitro. Pretreatment of the NF-κ B activation inhibitor attenuated the TGF-β ‑induced expression of β -catenin and differentiation in human lung fibroblasts. Similarly, LPA induced β-catenin expression in human lung fibroblasts, and pre-treatment of the neutralized anti-TGF-β antibody attenuated the LPA‑induced expression of β -catenin and differentiation in human lung fibroblasts. The results suggested that β-catenin expression is upregulated in lung fibroblast during differentiation, and that TGF-β induced β-catenin expression in human lung fibroblasts through the activation of NF-κ B. Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease characterized by distorted lung
Correspondence to: Dr Xia Sun, Department of Pediatrics, The University of Chicago, 5841 S Maryland Ave, Chicago, Illinois, IL 60637, USA E-mail: [email protected]
Dr Gang Wang, Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou, Jiangsu 223002, P.R. China E-mail: [email protected]
Key words: β-catenin, LPA, TGF-β, NF-κ B, pulmonary fibrosis, fibroblast
architecture and loss of respiratory function (1,2). Over five million individuals are afflicted with IPF in the USA, and the average survival time of IPF patients is only 2-5 years after initial diagnosis (3,4). Despite advances in understanding of the basic molecular pathways that drive this uncontrolled fibrotic process, lung transplantation remains the only effective cure for IPF (5). Thus, investigations to improve understanding of the pathological mechanisms and lead to the development of efficacious therapeutic approaches for IPF are necessary. Fibroblast accumulation leads to excessive scarring of lung tissue, and progressive and irreversible destruction of lung architecture, leading to loss of lung function, disruption of gas exchange and fibrogenesis in lung (6). Investigations thus far have demonstrated that transforming growth factor-β (TGF‑β), sphingosine 1 phospate (S1P) and lysophosphatidic acid (LPA) are involved in fibrogenesis in different organs, such as heart, liver and lung (4,6-9). Findings of recent studies suggest the crosstalk between TGF-β pathways and LPA or S1P pathways in lung fibroblasts (4,9). LPA also increases TGF-β expression and secretion in lung fibroblasts through the activation of its receptors (9). Additionally, in vitro investigations have shown that S1P induced the activation, differentiation and migration of lung fibroblasts (4). However, the crosstalk between the TGF- β pathway and other pathways on the effects in fibroblast differentiation remains to be determined. Wnt/β -catenin signaling is a key regulator in tissue repair, fibrosis, remodeling or destruction (2,10,11). Immuno histochemical staining shows the increase of nuclear β-catenin staining in IPF tissue sections (12,13). Unbiased microarray screens have revealed an increased expression of Wnt/β‑catenin target genes in lung tissue from IPF patients (12). Additionally, aberrant expression of extracellular matrix (ECM) proteins through epithelial-mesenchymal transition (EMT) and fibroblast differentiation contributes to pulmonary fibrosis (14-16). Mounting evidence suggest that the Wnt/β-catenin and TGF-β pathways contribute to the epithelial-mesenchymal transition (EMT) during pulmonary fibrogenesis (10,11,17,18). In vitro, upregulation of Wnt/β‑catenin signaling promotes the prolife ration of alveolar type II (AT2) cells and inhibits their ability to differentiate into alveolar type I (AT1 cells) cells (19). In lung fibroblasts, activation of Wnt/β -catenin signaling also stimulates ECM gene expression (20). However, the expression
1220
LI et al: β-CATENIN EXPRESSION IN LUNG FIBROBLAST
of β-catenin in lung fibroblasts has not been studied and the potential mechanism remains to be determined. In the present study, we investigated β-catenin expression under fibrotic conditions by western blot analysis. The results showed that β-catenin expression was markedly increased in lung tissue and fibroblasts from bleomycin‑challenged mice, and regulated by the activation of NF-κ B. Materials and methods Reagents and kits. Bleomycin sulfate was obtained from Hospira Inc. (Lake Forest, IL, USA), and neutralizing chicken anti-TGF-β1 antibody and control chicken IgG were obtained from R&D Systems (Minneapolis, MN, USA). Oleoyl lysophosphatidic acid (18:1 LPA) was obtained from Avanti Polar Lipids (Alabaster, AL, USA), and the cell lysis buffer was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Protease inhibitor cocktail tablets (EDTA-free Complete) were purchased from Roche Diagnostics (Indianapolis, IN, USA). Recombinant human TGF- β1 was obtained from Protech, Inc. (Rocky Hill, NJ, USA). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Rabbit anti-fibronectin and anti- β -catenin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-α-smooth muscle actin (α-SMA) and anti-β actin antibodies and Bay 11-7082 (NF-κ B inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Experimental pulmonary fibrosis model. The animal experiment of pulmonary fibrosis was designed as previously described (4,9). Briefly, C57/BL6 mice (male, aged 8 weeks) purchased from Jackson Laboratory (Bar Harbor, ME, USA) were used for bleomycin-induced fibrosis. Briefly, C57/BL6 mice were anesthetized (with a 3 ml/kg mixture of 25 mg/kg of ketamine in 2.5 ml of xylazine), followed by treatment with saline or bleomycin sulfate (1.5 U/kg of body weight, ~0.03 units/animal) in saline by an intratracheal injection in a total volume of 50 µl. Twenty-one days post‑bleomycin administration, the animals were sacrificed by cervical dislocation and the lungs were removed for histological staining and isolation of lung fibroblasts. Immunohistochemical staining and Masson's trichrome staining of mouse lung tissue. Lung tissues from mice with or without bleomycin challenge were embedded in paraffin and cut as 5-µm sections for staining. Following the removal of paraffin with xylene and clearing with alcohol, the slides were applied for immunohistochemical staining and Masson's trichrome staining and examined as previously described (9). Cell culture. Murine lung fibroblasts were isolated from mice with or without bleomycin (BLM) challenge as previously described (9,21). A human lung fibroblasts cell line (WI-38) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cells were grown and maintained in 6-well dishes with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Primary murine lung fibroblasts, isolated from C57/BL6 mice
with or without bleomycin treatment, were also cultured in DMEM containing 10% FBS. Treatment of neutralizing antibodies or NF- κ B inhibitor. Serum-starved (for 24 h) human lung fibroblasts (WI-38, ~90% confluence) were pretreated with neutralized anti-TGF- β antibody or control IgG antibody (5 µg/ml, 1 h). For nuclear factor κ-light-chain-enhancer of activated B cells (NF-κ B) inhibitor (Bay 11-7082), the compound was pretreated with a final concentration of 10 µM for 1 h (22). The cells were subsequently challenged with 18:1 LPA (10 µM) or TGF-β (5 ng/ml) for 48 h, and cell lysates (20 µg protein) were subjected to SDS-PAGE and western blotting. SDS-PAGE and western blotting. SDS-PAGE and western blotting were performed as previously described (9). The integrated density of pixels in each membrane was quantified using ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA, USA). Immunofluorescence staining. Immunofluorescence microscopy to determine protein expression was performed as previously described (9). Briefly, primary murine lung fibroblasts were grown on slide chambers for 24 h. The cells were fixed, incubated with primary antibodies (1:200 dilutions in blocking buffer) for 1 h and with Alexa Fluor secondary antibodies (1:200 dilutions in blocking buffer) for another 1 h, followed by mounting. The cells were then examined under a Nikon Eclipse TE 2000-S fluorescence microscope with a 60x oil immersion objective lens. Statistical analysis. Data are expressed as means ± SEM from at least three independent sets of experiments. Results were subjected to statistical analysis using one-way ANOVA or a two-tailed Student's t-test. P
Transforming growth factor β regulates β-catenin expression in lung fibroblast through NF-κB dependent pathway JIAN LI1, GANG WANG2 and XIA SUN3 1
Jinan Centre for Disease Control and Prevention, Jinan, Shandong 250001; 2Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou, Jiangsu 223002, P.R. China; 3 Department of Pediatrics, The University of Chicago, Chicago, IL 60637, USA Received March 7, 2014; Accepted August 8, 2014 DOI: 10.3892/ijmm.2014.1916
Abstract. β-catenin contributes to the pathogenesis of lung fibrosis. However, the expression of β -catenin in fibroblasts under fibrotic conditions has not been studied. We investigated the expression of β-catenin in lung fibroblasts from bleomycin (BLM)‑challenged mice and human lung fibroblasts treated with transforming growth factor β (TGF-β) or lysophosphatidic acid (LPA) by western blot analysis. The result showed that the expression of β-catenin was significantly increased in lung fibrotic foci and lung fibroblasts from bleomycin‑challenged mice. TGF- β stimulated β -catenin expression and induced differentiation in human lung fibroblasts in vitro. Pretreatment of the NF-κ B activation inhibitor attenuated the TGF-β ‑induced expression of β -catenin and differentiation in human lung fibroblasts. Similarly, LPA induced β-catenin expression in human lung fibroblasts, and pre-treatment of the neutralized anti-TGF-β antibody attenuated the LPA‑induced expression of β -catenin and differentiation in human lung fibroblasts. The results suggested that β-catenin expression is upregulated in lung fibroblast during differentiation, and that TGF-β induced β-catenin expression in human lung fibroblasts through the activation of NF-κ B. Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease characterized by distorted lung
Correspondence to: Dr Xia Sun, Department of Pediatrics, The University of Chicago, 5841 S Maryland Ave, Chicago, Illinois, IL 60637, USA E-mail: [email protected]
Dr Gang Wang, Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou, Jiangsu 223002, P.R. China E-mail: [email protected]
Key words: β-catenin, LPA, TGF-β, NF-κ B, pulmonary fibrosis, fibroblast
architecture and loss of respiratory function (1,2). Over five million individuals are afflicted with IPF in the USA, and the average survival time of IPF patients is only 2-5 years after initial diagnosis (3,4). Despite advances in understanding of the basic molecular pathways that drive this uncontrolled fibrotic process, lung transplantation remains the only effective cure for IPF (5). Thus, investigations to improve understanding of the pathological mechanisms and lead to the development of efficacious therapeutic approaches for IPF are necessary. Fibroblast accumulation leads to excessive scarring of lung tissue, and progressive and irreversible destruction of lung architecture, leading to loss of lung function, disruption of gas exchange and fibrogenesis in lung (6). Investigations thus far have demonstrated that transforming growth factor-β (TGF‑β), sphingosine 1 phospate (S1P) and lysophosphatidic acid (LPA) are involved in fibrogenesis in different organs, such as heart, liver and lung (4,6-9). Findings of recent studies suggest the crosstalk between TGF-β pathways and LPA or S1P pathways in lung fibroblasts (4,9). LPA also increases TGF-β expression and secretion in lung fibroblasts through the activation of its receptors (9). Additionally, in vitro investigations have shown that S1P induced the activation, differentiation and migration of lung fibroblasts (4). However, the crosstalk between the TGF- β pathway and other pathways on the effects in fibroblast differentiation remains to be determined. Wnt/β -catenin signaling is a key regulator in tissue repair, fibrosis, remodeling or destruction (2,10,11). Immuno histochemical staining shows the increase of nuclear β-catenin staining in IPF tissue sections (12,13). Unbiased microarray screens have revealed an increased expression of Wnt/β‑catenin target genes in lung tissue from IPF patients (12). Additionally, aberrant expression of extracellular matrix (ECM) proteins through epithelial-mesenchymal transition (EMT) and fibroblast differentiation contributes to pulmonary fibrosis (14-16). Mounting evidence suggest that the Wnt/β-catenin and TGF-β pathways contribute to the epithelial-mesenchymal transition (EMT) during pulmonary fibrogenesis (10,11,17,18). In vitro, upregulation of Wnt/β‑catenin signaling promotes the prolife ration of alveolar type II (AT2) cells and inhibits their ability to differentiate into alveolar type I (AT1 cells) cells (19). In lung fibroblasts, activation of Wnt/β -catenin signaling also stimulates ECM gene expression (20). However, the expression
1220
LI et al: β-CATENIN EXPRESSION IN LUNG FIBROBLAST
of β-catenin in lung fibroblasts has not been studied and the potential mechanism remains to be determined. In the present study, we investigated β-catenin expression under fibrotic conditions by western blot analysis. The results showed that β-catenin expression was markedly increased in lung tissue and fibroblasts from bleomycin‑challenged mice, and regulated by the activation of NF-κ B. Materials and methods Reagents and kits. Bleomycin sulfate was obtained from Hospira Inc. (Lake Forest, IL, USA), and neutralizing chicken anti-TGF-β1 antibody and control chicken IgG were obtained from R&D Systems (Minneapolis, MN, USA). Oleoyl lysophosphatidic acid (18:1 LPA) was obtained from Avanti Polar Lipids (Alabaster, AL, USA), and the cell lysis buffer was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Protease inhibitor cocktail tablets (EDTA-free Complete) were purchased from Roche Diagnostics (Indianapolis, IN, USA). Recombinant human TGF- β1 was obtained from Protech, Inc. (Rocky Hill, NJ, USA). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Rabbit anti-fibronectin and anti- β -catenin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-α-smooth muscle actin (α-SMA) and anti-β actin antibodies and Bay 11-7082 (NF-κ B inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Experimental pulmonary fibrosis model. The animal experiment of pulmonary fibrosis was designed as previously described (4,9). Briefly, C57/BL6 mice (male, aged 8 weeks) purchased from Jackson Laboratory (Bar Harbor, ME, USA) were used for bleomycin-induced fibrosis. Briefly, C57/BL6 mice were anesthetized (with a 3 ml/kg mixture of 25 mg/kg of ketamine in 2.5 ml of xylazine), followed by treatment with saline or bleomycin sulfate (1.5 U/kg of body weight, ~0.03 units/animal) in saline by an intratracheal injection in a total volume of 50 µl. Twenty-one days post‑bleomycin administration, the animals were sacrificed by cervical dislocation and the lungs were removed for histological staining and isolation of lung fibroblasts. Immunohistochemical staining and Masson's trichrome staining of mouse lung tissue. Lung tissues from mice with or without bleomycin challenge were embedded in paraffin and cut as 5-µm sections for staining. Following the removal of paraffin with xylene and clearing with alcohol, the slides were applied for immunohistochemical staining and Masson's trichrome staining and examined as previously described (9). Cell culture. Murine lung fibroblasts were isolated from mice with or without bleomycin (BLM) challenge as previously described (9,21). A human lung fibroblasts cell line (WI-38) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cells were grown and maintained in 6-well dishes with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Primary murine lung fibroblasts, isolated from C57/BL6 mice
with or without bleomycin treatment, were also cultured in DMEM containing 10% FBS. Treatment of neutralizing antibodies or NF- κ B inhibitor. Serum-starved (for 24 h) human lung fibroblasts (WI-38, ~90% confluence) were pretreated with neutralized anti-TGF- β antibody or control IgG antibody (5 µg/ml, 1 h). For nuclear factor κ-light-chain-enhancer of activated B cells (NF-κ B) inhibitor (Bay 11-7082), the compound was pretreated with a final concentration of 10 µM for 1 h (22). The cells were subsequently challenged with 18:1 LPA (10 µM) or TGF-β (5 ng/ml) for 48 h, and cell lysates (20 µg protein) were subjected to SDS-PAGE and western blotting. SDS-PAGE and western blotting. SDS-PAGE and western blotting were performed as previously described (9). The integrated density of pixels in each membrane was quantified using ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA, USA). Immunofluorescence staining. Immunofluorescence microscopy to determine protein expression was performed as previously described (9). Briefly, primary murine lung fibroblasts were grown on slide chambers for 24 h. The cells were fixed, incubated with primary antibodies (1:200 dilutions in blocking buffer) for 1 h and with Alexa Fluor secondary antibodies (1:200 dilutions in blocking buffer) for another 1 h, followed by mounting. The cells were then examined under a Nikon Eclipse TE 2000-S fluorescence microscope with a 60x oil immersion objective lens. Statistical analysis. Data are expressed as means ± SEM from at least three independent sets of experiments. Results were subjected to statistical analysis using one-way ANOVA or a two-tailed Student's t-test. P
